TLR22-mediated activation of TNF-α-caspase-1/IL-1β inflammatory axis leads to apoptosis of Aeromonas hydrophila-infected macrophages

TLR22-Induced Pro-Apoptotic mtROS Abets UPRmt-Mediated Mitochondrial Fission in Aeromonas hydrophila-Infected Headkidney Macrophages of Clarias gariepinus

“…The fish were euthanized using MS 222 (Sigma), headkidney excised aseptically and HKM were isolated using 34/51% percoll gradient as described earlier ( 33 ). The HKM were infected with A. hydrophila (MOI 1:50) for 1 h and extracellular bacteria was removed using chloramphenicol (30 µg/mL) as described earlier ( 16 ).…”

“…The HKM were pre-incubated separately with mPTP inhibitor [Cyclosporin A (CsA), 5 µM, Sigma], mitochondrial Ca 2+ uniporter (MCU) inhibitor [Ruthenium Red (RR), 20 µM, Sigma], caspase-9 inhibitor [Z-LEHD-FMK, 7.5 µM, Sigma], DRP-1 inhibitor [Mdivi-1, 25 µM, Sigma], HIF-1α inhibitor [Dimethyl-bisphenol A (di-BPA), 200 µM, Abcam], mtROS inhibitor [YCG063, 10 µM, Calbiochem], caspase-9 inhibitor [Z-LEHD-FMK, 7.5 µM, Biovision], caspase-3 inhibitor [Ac-DEVD-CHO, 10 µM, Sigma], for 1 h and then infected with A. hydrophila as mentioned earlier ( 16 ). The inhibitor concentrations used in the study had no adverse effects on the viability of HKM and bacterial growth per se (data not shown).…”

“…The total RNA was isolated using TRI reagent (MRC) as per the manufacturer’s instructions. The cDNA was prepared using Revert Aid First Strand cDNA synthesis (Thermo Fischer Scientific) from 1 µg of DNase-treated RNA as described earlier ( 16 ).…”

“…Gene expression studies were performed using ViiA Real-Time PCR system (ABI) and SYBR green PCR Master Mix (ABI) with specific primers ( Table 2 ). The expression of the genes was quantitated by comparative ΔΔC T method and normalized against β-actin (housekeeping gene) as described earlier ( 16 ).…”

“…Previous studies have implicated the role of TLR22 in the fish immune response against microbial infection ( 12 – 15 ). Recently, we reported the anti-bacterial and pro-apoptotic role of TLR22 in fish macrophages ( 16 ) but how it contrives innate immune signaling pathways in fish needs to be investigated. Although TLR signaling has been implicated in mtROS production ( 5 ), but the primal role of TLR22 has not been reported.…”